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Keywords:

  • isoelectric point;
  • electric charge of proteins;
  • acid–base residues;
  • PICAL

Abstract

  1. Top of page
  2. Abstract
  3. DETERMINATION OF THE ISOELECTRIC POINT (pI) VALUE OF PROTEINS BY THE SIMPLIFIED METHOD
  4. DETERMINATION OF pI VALUE AND ELECTRIC CHARGES OF PROTEINS VERSUS pH USING THE IMPROVED OSCILLATING METHOD (IOM)
  5. GENERAL CONSIDERATIONS ON THE pI VALUES OF THE PROPOSED PROTEINS
  6. ON THE NOMENCLATURE OF THE ACID–BASE RESIDUES USED IN SOME TEXTBOOKS OF BIOCHEMISTRY
  7. Acknowledgements
  8. REFERENCES

The main object of this work is to present the pedagogical usefulness of the theoretical methods, developed in this laboratory, for the determination of the isoelectric point (pI) and the net electric charge of proteins together with some comments on the naming of the acid-base residues of proteins

In the recent years, we have approached the theoretical calculation of the isoelectric point and the net electric charge of proteins and other macromolecules [1–7] and we have used this study to explain these topics to students in regular academic courses of Biochemistry. The object of this publication is to present our pedagogical experience in this field, new applications and possibilities of using the Internet for these procedures and some comments on the naming of the acid–base residues of proteins, widely used in textbooks of Biochemistry.

An essential point for the development of this work was to rename the acid–base residues of proteins as type-N groups (from Neutral when undissociated) and type-P groups (from Positively charged when undissociated). A full classification of these acid–base residues as type-P or type-N groups (or subgroups), and their pK values is presented in Table I.

Table I. Amino acid residues potentially bearing electric charges
Group typeConjugate acidConjugate base + H+pK Value
  1. The acid-base residues are classified as belonging to group-N or -P depending on whether they are neutral (N) or positively (P) charged when undissociated.

N1R-COOH (terminal carboxyl)R-(COO) (terminal carboxylate)3.2
N2R-COOH (β-carboxyl of aspartate)R-(COO) (β-carboxylate of aspartic)4.0
N3R-COOH (γ-carboxyl of glutamate)R-(COO) (γ -carboxylate of glutamic)4.5
N4R-SH (thiol of cysteine)R-S (thiolate of cysteine)9.0
N5R-C6H4OH (phenyl of tyrosine)R-(C6H4O) (phenolate of tyrosine)10
NaNa= [N1 or N2 or N3] 4.2
NbNb = [N4 or N5] 9.5
P1R-(C3H5N2)+ (imidazolium of histidine)R-C3H4N2 (imidazole of histidine)6.4
P2R-(NH3)+ (terminal ammonium)R-NH2 (terminal amino)8.2
P3R-(NH3)+ (ε-ammonium of lysine)R-NH2 (ε-amino of lysine)10.4
P4R-(CH6N3)+ (guanidinium of arginine)R-CH5N3 (guanidine of arginine)12
PaPa = [ P2 or P3 or P4] 11.2
PbPb = P1 6.4

There are a plethora of online methods for the determination of the isoelectric point of proteins, some of which can be used freely from the Internet:

However, none of these applications is intended as a teaching tool to address the concept of protein pI or the relationships between the pI value of a protein and the pK values of its acid–base groups. An exception is a report recently made available at http://isoelectric. ovh.org, which pursues objectives somewhat similar to ours.

The following is an application of two useful methods developed by us for the determination of the pI value and electric charge of a protein. The Simplified Method, calculates the pI value with the help of three Tables included in this article, and without assistance of a computer. The other, Improved Oscillating Method (IOM), can be run with a program freely accessible from the Web. The IOM has been adapted for student use and has some special characteristics. The pKa values of amino acids can be easily modified and the pI values compared with those obtained with the standard pK values of amino acids. In addition, a list of specially designed proteins with didactical purposes and with a predetermined amino acid composition is included. Finally, a graph with the charge of the protein vs. the pH values can be easily obtained.

DETERMINATION OF THE ISOELECTRIC POINT (pI) VALUE OF PROTEINS BY THE SIMPLIFIED METHOD

  1. Top of page
  2. Abstract
  3. DETERMINATION OF THE ISOELECTRIC POINT (pI) VALUE OF PROTEINS BY THE SIMPLIFIED METHOD
  4. DETERMINATION OF pI VALUE AND ELECTRIC CHARGES OF PROTEINS VERSUS pH USING THE IMPROVED OSCILLATING METHOD (IOM)
  5. GENERAL CONSIDERATIONS ON THE pI VALUES OF THE PROPOSED PROTEINS
  6. ON THE NOMENCLATURE OF THE ACID–BASE RESIDUES USED IN SOME TEXTBOOKS OF BIOCHEMISTRY
  7. Acknowledgements
  8. REFERENCES

As previously reported [1–8], the electric charge of type-N and type-P groups at a given pH value can be calculated according to equations 1 and 2, both derived from the Henderson-Hasselbach equation:

  • equation image(1)
  • equation image(2)

By application of these equations to a macromolecule containing a number of acid–base groups, of known pK values, its isoelectric point can be determined. In a previous work [2], three methods for the theoretical determination of the pI values of proteins were described: Simplified, Abridged and Comprehensive. The Abridged [2] and the Comprehensive [2, 3, 5] methods convey the use of complex mathematical calculations and will not be considered here. A short presentation of the Simplified Method [2] is given below, because of its Simplicity and potential use by students, without any need of computer help. This method requires only the handling of Tables II–IV. The rationale of this method is partially based on the following premises [2]: (i) due to the similarity in their pK values, N1, N2, and N3 are set equal to Na (pKNa value of 4.2); N4 and N5 set equal to Nb (pKNb value of 9.5); P2, P3, and P4 set equal to Pa (pKPa value of 11.2); by analogy, P1 is renamed as Pb (Table I); the same simplification on the pK values was applied in the Abridged Method [2] and, in this context, the results obtained with the Simplified and Abridged methods are rather similar; (ii) the relationship between the number of type-Pa and type-Na groups (Pa/Na or Na/Pa) is the main factor influencing the pI value of a protein (see also below); (iii) at some pH ranges (5–7), the charge values of the Pa and the Na groups are considered with opposite charge, and the charge afforded by the Nb groups negligible. The theoretical basis of the Simplified Method is described in detail in [2]. Following the information given in Tables II–IV, students may calculate the ratios involving the number of Na, Nb, Pa and Pb groups present in a protein and from those ratios its pI value can be easily deduced. Depending on the time available in the class room, the professor could invite the students to apply Tables II–IV for the determination of the pI value of a certain protein, go through the theoretical bases or encourage the students to peruse the original paper [2].

Table II. A simplified procedure to calculate approximate pI values of proteins (modified from [2])
CaseProcedure
  1. Na = 1 + Asp + Glu; Pa = 1 + Lys + Arg; Nb = Cys + Tyr; Pb = His. If Na > Pa, apply c1, c2; if Pa > Na, apply c3, c4.

C1Na/(Pa + Pb) ≥ 1.06 [RIGHTWARDS ARROW] Table III
C2Na/(Pa + Pb) < 1.06 [RIGHTWARDS ARROW] Calculate Pb/(Na − Pa) [RIGHTWARDS ARROW] Table IV
C3Pa/(Na + Nb) ≥ 1.09 [RIGHTWARDS ARROW] Table III
C4Pa/(Na + Nb) < 1.09 [RIGHTWARDS ARROW] Calculate Nb/(Pa − Na) [RIGHTWARDS ARROW] Table IV
Table III. Correlation between the pI value of a protein and its content in aspartate, glutamate, lysine, and arginine (modificated from [2])
pINa/PaPa/NapI
  1. Na = 1 + Asp + Glu; Pa = 1 + Lys + Arg.

4.02.58492.584911.4
4.12.25892.258911.3
4.22.00002.000011.2
4.31.79431.794311.1
4.41.63101.631011.0
4.51.50121.501210.9
4.61.39811.398110.8
4.71.31621.316210.7
4.81.25121.251210.6
4.91.19951.199510.5
5.01.15851.158510.4
5.11.12591.125910.3
5.21.10001.100010.2
5.31.07941.079410.1
5.41.06311.063110.0
5.51.05011.05019.9
5.61.03981.03989.8
5.71.03161.03169.7
5.81.02511.02519.6
5.91.01991.01999.5
6.01.01581.01589.4
6.11.01261.01269.3
6.21.01001.01009.2
6.31.00791.00799.1
6.41.00631.00639.0
6.51.00501.00508.9
6.61.00401.00408.8
6.71.00311.00318.7
6.81.00251.00258.6
6.91.00191.00198.5
7.01.00151.00158.4
7.11.00121.00128.3
7.21.00091.00098.2
7.31.00071.00078.1
7.41.00051.00058.0
7.51.00031.00037.9
7.61.00011.00017.8
7.71.00001.00007.7
Table IV. Correlation between pI values of proteins and some ratios of their acid-base groups (modified from [2])
pIPb/(Na − Pa)Nb/(Pa − Na)pI
  1. Na = 1 + Asp + Glu; Pa = 1 + Lys + Arg; Nb = Cys + Tyr; Pb = His.

5.21.0631
5.31.0794
5.41.1000
5.51.1259
5.61.1585
5.71.19951.199510.2
5.81.25121.251210.1
5.91.31621.316210.0
6.01.39811.39819.9
6.11.50121.50129.8
6.21.63101.63109.7
6.31.79431.79439.6
6.42.00002.00009.5
6.52.25892.25899.4
6.62.58492.58499.3
6.72.99532.99539.2
6.83.51193.51199.1
6.94.16234.16239.0
7.04.98114.98118.9
7.16.01196.01198.8
7.27.30967.30968.7
7.38.94338.94338.6
7.411.000011.00008.5
7.513.589313.58938.4
7.616.848916.84898.3
7.720.952620.95268.2
7.826.118926.11898.1
7.932.622832.62288.0
8.040.810740.81077.9
8.151.118751.11877.8
8.264.095764.09577.7
8.380.432880.43287.6
8.4101.000101.00007.5

DETERMINATION OF pI VALUE AND ELECTRIC CHARGES OF PROTEINS VERSUS pH USING THE IMPROVED OSCILLATING METHOD (IOM)

  1. Top of page
  2. Abstract
  3. DETERMINATION OF THE ISOELECTRIC POINT (pI) VALUE OF PROTEINS BY THE SIMPLIFIED METHOD
  4. DETERMINATION OF pI VALUE AND ELECTRIC CHARGES OF PROTEINS VERSUS pH USING THE IMPROVED OSCILLATING METHOD (IOM)
  5. GENERAL CONSIDERATIONS ON THE pI VALUES OF THE PROPOSED PROTEINS
  6. ON THE NOMENCLATURE OF THE ACID–BASE RESIDUES USED IN SOME TEXTBOOKS OF BIOCHEMISTRY
  7. Acknowledgements
  8. REFERENCES

Theoretical Bases

The Improved Oscillating Method (IOM) is written in Visual Basic [6, 7] without the mathematical complications of the Abridged [2] and the Comprehensive Methods [2, 3, 5]. It can be used not only to calculate the pI value of a protein, with the same accuracy as the Comprehensive Method, but also Evaluate the electric charge of a protein at different pH values. The Improved Oscillating Method (IOM) [6, 7], has been partially rewritten and adapted for pedagogical purposes and has been applied to the software “PICAL for BAMBED” (pI Calculation for Biochemistry and Molecular Biology Education).

The core of the program uses, by default, the standard pK values of each one of the acid–base groups of proteins (Table I). After introducing the number of each one of these groups, the program uses equations 1 and 2 to calculate the charge of each residue (N1, N2, N3, N4, N5, P1, P2, P3, P4, Table I) and to estimate the net charge of the protein (NQ) at increasing pH values, starting from zero and with increments of 0.1 pH units. Note as all the natural proteins are positively charged (NQ > 0) at very acid pH values, when the type-N and the type-P groups are neutral or positively charged, respectively; depending on the nature and content of its type-N groups, the charge of the protein becomes gradually less positive, as the pH value of the medium increases, until a value of net charge NQ ≤ 0 is reached. The pH value corresponding to the transition from NQ > 0 to NQ < 0 is called pI(a). In the very improbable case that NQ(a) is exactly zero, the pI for the protein [pI = pI(a)] is reported on the screen. When NQ(a) < 0, the following situation arises: at pH = pI(a) the net charge of the protein is negative and at pH = pI(a) − 0.1 the charge is positive. Therefore pI(a) is the protein pI value with one significant decimal figure. For the calculation of the second decimal figure of pI, the loop starts at pH = pI(b) = p(a) − 0.1, with increments of 0.01 pH units. A pH value pI(c) is then reached at which NQ(c) ≤ 0. The process continues oscillating until the precision required is reached. The number of decimal figures of the pI value can be set up by the operator (see below).

The algorithm was first developed for handling only one protein (Oscillating Method) [6] and later adapted (Improved Oscillating Method) for the calculation of the net electric charge and pI values of the proteins contained in a file or data base [7]. Computer details of both procedures have been described in [6, 7]. What follows is a simple route to facilitate its operation together with some significant examples to be used by students in a regular course of Biochemistry and/or by researchers in basic or applied research on proteins.

Model Proteins

Several theoretical proteins were designed for student analysis, with compositions chosen to give an overview on the influence of each type of amino acid on the pI of a protein. They are listed in www.estudiantesmedicina. com/bambed.htm and in an abbreviated form in Figs. 1–4. These proteins have been grouped in several databases (A, A1, A2, A3, B, B1, B2, and B3) with the characteristics and purposes described below.

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Figure 1. Amino acid composition of files A and B, containing only type-N (Asp and Glu) and type-P residues (Lys and Arg) with different Na and Pa ratios. The pI values obtained with the IOM method are related to the ratio Na and Pa in the upper part of the Figure.

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Figure 2. Amino acid composition of files A1 and B1, containing type-N (Asp and Glu), and type-P residues (Lys and Arg) with different Na and Pa ratios and different amounts of His, as indicated. The pI values obtained with the IOM method are refered to the ratio Na and Pa in the upper part of the Figure.

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Figure 3. Amino acid composition of files A2 and B2, containing type-N (Asp and Glu), and type-P residues (Lys and Arg) with different Na and Pa ratios and different amounts of Tyr, as indicated. The pI values obtained with the IOM method are refered to the ratio Na and Pa in the upper part of the Figure.

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Figure 4. Amino acid composition of files A3 and B3, containing type-N (Asp and Glu), and type-P residues (Lys and Arg) with different Na and Pa ratios and different amounts of Cys, as indicated. The pI values obtained with the IOM method are refered to the ratio Na and Pa in the upper part of the Figure.

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Proteins with Only Type-Na and Type-Pa Acid–Base Groups (Contained in Files A and B)—

The proteins containing both, but only, Na groups (aspartates + glutamates) and Pa groups (lysines + arginines) have been grouped in files A and B. File A contains proteins with a variable number of aspartates (10–42) and glutamates (10–42) and a fixed number of lysines (10) and arginines (10), which makes a total of 33 proteins, all of them with a ratio Na/Pa ≥ 1 (Fig. 1, file A; see previous page). File B contains proteins (with a ratio Pa/Na ≥ 1) with a variable number of lysines (10–42) and arginines (10–42) and a fixed number of aspartates (10) and glutamates (10), also making a total of 33 proteins (Fig. 1, file B).

Influence of the Incorporation of Histidines, Cysteines, or Tyrosines to Proteins from File A and File B—

The influence of these amino acids on the pI value of proteins containing only aspartates, glutamates, lysines and arginines was approached in a similar way by adding histidines, cysteines or tyrosines residues. Each one of the 33 proteins present in file A and file B were supplemented with series of 0, 1, 5, 10, or 15 histidines (Fig. 2, files A1 and B1); 0, 1, 5, 10, or 15 tyrosines (Fig. 3, files A2, B2); 0, 1, 5, 10, or 15 cysteines (Fig. 4, files A3, B3). Each file A1, A2, A3, B1, B2, B3, contains 33 × 5 = 165 proteins.

Calculation of the Net Electric Charge and pI Value of a Protein—

The first screen of the program (Fig. 5) presents three choices: to calculate the isoelectric point and charge of one protein, to load a database or to create a new database. If the first option is selected the second screen “Calculation of the isoelectric point of one protein” appears (Fig. 6). The name of the protein (optional) and the number of each one of the amino acids (potentially) bearing charge is then requested. The program assumes, by default, the occurrence of one terminal carboxylic and one terminal ammonium group in the protein. The number of decimal figures to be reported for the isoelectric point may also be set. After pressing the “Calculation” button, the isoelectric point value and a graph showing the net electric charge of the protein vs. pH appears on the screen. The following possibilities are now open: i) to produce a Table of the electric charge of the protein at each pH value; ii) to save the values obtained for this protein; (iii) to load a new protein (human hemoglobin α chain is incorporated in the program as an example); (iv) to change the pK value of any amino acid and to proceed to a new calculation; in this case the influence of changing the pK values of one or several acid–base residues on the electric charge and the pI value of one protein can be graphically visualized. The original pK values of the amino acids can be restored with the buttons “change pK values/reset values”; the original pK values of the amino acids are restored every time the program is initiated. With this modality, the parameters of each protein can be determined sequentially.

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Figure 5. The first screen of PICAL invites to follow three options: work with one protein, with a stored database or to create a new database (modified from [7]).

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Figure 6. Second main screen of PICAL: calculation of the electric charge and pI value of one protein. For more details see the text (modified from [7]).

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Calculation of the Net Electric Charge and pI Values of Several Proteins or Proteins from a Data Bank—

After pressing the “Load database” button in screen number 1 (Fig. 5), the files stored by default appear. These files (A, A1, A2, A3, B, B1, B2, and B3) correspond to the pool of proteins described above. The abbreviated amino acid composition of these files is in Figs. 1–4, and the complete composition can be found in www.estudiantesme dicina.com/bambed.htm. After selecting any of these files, screen number 3 (Fig. 7) is displayed offering multiple options. By default, the first protein of the chosen file appears, with the number of amino acids and pI already calculated. The net electric charge vs. pH chart of this particular protein can be seen on the screen by pressing the corresponding button. By pressing the First, Back, Next, and Last buttons, several proteins from the selected file can be handled.

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Figure 7. Third main screen of PICAL: calculation of the electric charge and pI value from a database. For more details see the text (modified from [7]).

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Additionally, the following operations can be executed jointly with the pool of proteins contained in a file: determination of the pI value of all the proteins of the file; ordering of the proteins according to their names (by default), pI value and data input and, once ordered, a new global pI chart and list of the proteins can be obtained; change of a file by deletion or addition of new proteins; loading and creation of new data base are also possible. This is made directly from the program (using the buttons “Load Database” and “Create New Database”, Fig. 5), which use the file format *.mdb (Microsoft Access). Other uses of the program, including “global pI charge”, “pH/charge chart (for one protein)”, “go back”, “exit” or “print” buttons, are self-evident.

GENERAL CONSIDERATIONS ON THE pI VALUES OF THE PROPOSED PROTEINS

  1. Top of page
  2. Abstract
  3. DETERMINATION OF THE ISOELECTRIC POINT (pI) VALUE OF PROTEINS BY THE SIMPLIFIED METHOD
  4. DETERMINATION OF pI VALUE AND ELECTRIC CHARGES OF PROTEINS VERSUS pH USING THE IMPROVED OSCILLATING METHOD (IOM)
  5. GENERAL CONSIDERATIONS ON THE pI VALUES OF THE PROPOSED PROTEINS
  6. ON THE NOMENCLATURE OF THE ACID–BASE RESIDUES USED IN SOME TEXTBOOKS OF BIOCHEMISTRY
  7. Acknowledgements
  8. REFERENCES

By application of the Improved Oscillating Method to the proteins of the files described above, Figs. 1–4 were obtained. As shown in Fig. 1 the pI value of a protein is greatly influenced by the ratio of its content in aspartates and glutamates and of lysines and arginines, i.e. the ratios Na/Pa and Pa/Na: that one giving a value of ≥ 1 is used in the graphs. A ratio of 1 corresponds to a pI value of 6.89. The ratios between Pa and Na have been calculated considering also the presence of one terminal carboxyl and one terminal ammonium group in a protein. The pI value of a protein increases, as the ratio Pa/Na increases and decreases as the ratio Na/Pa increases (Fig. 1).

The addition of histidines to proteins with a ratio Na/Pa ≥ 1 tends to increase their pI values (Fig. 2, A1) and have almost no influence on proteins with a ratio Pa/Na ≥ 1 (Fig. 2, B1). Contrary to histidine, the addition of tyrosines (Fig. 3, A2, B2) or cysteines (Fig. 4, A3, B3) has more influence on the pI values of proteins with a Pa/Na ≥ 1). For a discussion on these points see [2].

To better appreciate the influence of the addition of histidines, tyrosines or cysteines on the pI value of the standard proteins composed only by Pa and Na groups, the curves of the pI values of the basal proteins (Fig. 1, A and B) have been superimposed, when appropriate, as dashed lines in the graphs in Figs. 2–4.

Comparison of the Simplified and the Improved Oscillating Methods

As stated above the Simplified (and Abridged) Method is based on the main premise that a mean pK value was assumed for some groups of residues: [N1 or N2 or N3] = Na (pKNa value = 4.2); [N4 or N5] = Nb (pKNb = 9.5); [P2 or P3 or P4 ] = Pa (pKPa = 11.2). The possibility offered by the Improved Oscillating Method of changing the standard pK value of any acid–base residue of a protein allows an examination of the deviation from the pI value and net electric charge of proteins introduced by the Simplified (or Abridged) Method [2] in comparison with IOM. This was made by running the IOM, with the pK values (Table I) used in the Simplified Method. As an example, the charts of the pI value of proteins contained in files A, B (the basal proteins) and A1, B1 (the basal proteins supplemented with histidine) are shown in Fig. 8. The results presented in Fig. 8 show that the pI values obtained by the Simplified Method using the Tables II–IV are very similar to those obtained with IOM.

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Figure 8. Representation of the proteins from files A, B, A1 and B1, with the IOM (Improved Oscillating Method), using the standard pK values (continuous line) or the pK values used in the Simplified/Abridged Method [2, 7] (discontinuous line). The amino acid composition of those files are in Figs. 1 and 2. For more details see the text.

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Other Pedagogical Suggestions

The Improved Oscillating Method, here adapted for its use in regular courses of Biochemistry, offers many alternatives to existing methods. Its use may stimulate the students to investigate the electric charge and pI values of proteins, including those devised by themselves, to measure the influence of changing pK values, or to test the effect of covalent modifications of proteins (such as phosphorylations) on both parameters. The deviation of the experimental value from the theoretical pI value of a protein may indicate changes in the pK values of some acid–base residues in the native protein. The IOM may serve as a predictive tool to determine the best chromatographic conditions (type of resins and pH to be used) or the best pH for the precipitation of a protein (pH = pI). The IOM method (used by PICAL program) can be easily extended for use in the determination of the electric charge of nucleosides, nucleotides and polynucleotides [1] and to devise and understand a chromatographic method for their separation. In our view the theoretical methods here described for the determination of the electric charge and pI value of macromolecules offer many alternatives for their use in the classroom, or in the laboratory, to stimulate students' participation. In this sense, we developed a written contest around this methodology, with strong participation, among the students of regular courses of Biochemistry. The students were invited to analyze the pI value and electric charge of model proteins designed by them, with a brief discussion on the results obtained. At the end of the contest three diplomas (first, second and third) were awarded in a public ceremony.

ON THE NOMENCLATURE OF THE ACID–BASE RESIDUES USED IN SOME TEXTBOOKS OF BIOCHEMISTRY

  1. Top of page
  2. Abstract
  3. DETERMINATION OF THE ISOELECTRIC POINT (pI) VALUE OF PROTEINS BY THE SIMPLIFIED METHOD
  4. DETERMINATION OF pI VALUE AND ELECTRIC CHARGES OF PROTEINS VERSUS pH USING THE IMPROVED OSCILLATING METHOD (IOM)
  5. GENERAL CONSIDERATIONS ON THE pI VALUES OF THE PROPOSED PROTEINS
  6. ON THE NOMENCLATURE OF THE ACID–BASE RESIDUES USED IN SOME TEXTBOOKS OF BIOCHEMISTRY
  7. Acknowledgements
  8. REFERENCES

For the sake of simplicity we shall consider firstly the carboxyl and ammonium groups. At a first glance, the R-COOH (carboxyl) group of any amino acid or protein is an acidic group, able to donate protons, once the pH is above 3–4. Considering a physiological pH value (around 7.4) this group is in the form of R-COO (carboxylate), a conjugate base able to take protons from the medium. By the same token, the R-NH2 (amino) residue is a basic group able to take protons. However in a very wide range of pH values (from 0 to 9) this group is in the form of R-NH3+ (ammonium), an acid able to donate protons to the medium. At physiological pH values, the R-COO and R-NH3+ groups could be theoretically regarded as basic and acidic residues, respectively, in contrast to their usual treatment in many textbooks. Similar reasoning could be applied to other acid–base groups of amino acids and proteins (see Table I). Because of the difficulty in qualifying those groups as acidic or basic residues, we started considering all these groups in the undissociated form, as belonging to type-N, type-P, or to any of their subgroups (see above; Table I).

Certain confusion may then arise in the naming of the acid–base residues of amino acids. The plethora of excellent textbooks for Biochemistry and the fact that the same textbook frequently applies distinct names to those residues depending on the framework in which they are discussed open so many possibilities that it is out of the scope of this article to present a survey of them. Nevertheless, as these relate the electric charge of proteins, a small summary is below presented.

In general, there is a tendency to consider aspartic and glutamic acid as acidic amino acids carrying a negative charge and to consider lysine, arginine and histidine as basic amino acids carrying a positive charge [9–14]. In some cases amino acids are mainly classified as uncharged or charged [14, 15]. Finally the presence of protonated species (HA and R-NH3+) and unprotonated species (A and R-NH2) are clearly stated in some texts [14, 16].

In our view, it would be convenient to state that each one of those residues, potentially bearing charge, behaves as an acid–base pair, in the Brönsted-Lowry theory, in which depending on the pH, the acid–conjugate or the basic-conjugate forms predominate (Table I).

  • 1
    At acidic pH values (<3) all the acid–base residues of the amino acids are in the acidic form and have either a neutral charge (N-groups) or a positive charge (P groups) (Table I).
  • 2
    At a physiological pH value (7.4), the acid–base residues are: a) in their conjugate base form, with a negative charge (R-COO), mainly uncharged (imidazole of histidine), or b) in their acidic form, bearing no charge (phenyl of tyrosine and thiol of cysteine) or a positive charge (ammonium of lysine and guanidinium of arginine) (Table I).
  • 3
    At more basic pH values (>12) all the amino acids are predominantly in their conjugate base form.

It seems to us that given the diversity of acid–base forms present at physiological pH it could be more convenient to pivot the nomenclature at acid pH values. Disregarding other acid–base residues, the amino acids could be classified as monoammonium monocarboxylic (1:1 ammonium:carboxylate); diammonium monocarboxylic (2:1 ammonium:carboxylate) and monoammonium dicarboxylic (1:2 ammonium:carboxylate) depending on the relative content of these two types of groups, so determinants in the charge of the proteins.

Acknowledgements

  1. Top of page
  2. Abstract
  3. DETERMINATION OF THE ISOELECTRIC POINT (pI) VALUE OF PROTEINS BY THE SIMPLIFIED METHOD
  4. DETERMINATION OF pI VALUE AND ELECTRIC CHARGES OF PROTEINS VERSUS pH USING THE IMPROVED OSCILLATING METHOD (IOM)
  5. GENERAL CONSIDERATIONS ON THE pI VALUES OF THE PROPOSED PROTEINS
  6. ON THE NOMENCLATURE OF THE ACID–BASE RESIDUES USED IN SOME TEXTBOOKS OF BIOCHEMISTRY
  7. Acknowledgements
  8. REFERENCES

The authors thank Dr. María Antonia Günther for critical reading of the manuscript. Part of this investigation was carried out during the stay of A. Maldonado in the Plastic Surgery Department, Unfallkrankenhaus Berlin (UKB), Germany, and they also thank Prof. Dr. Markus Küntscher for his encouragement and support. A. Maldonado is actually a MIR (Medical Resident) in the Plastic Surgery Department of the Hospital Universitario de Getafe (Madrid, Spain).

REFERENCES

  1. Top of page
  2. Abstract
  3. DETERMINATION OF THE ISOELECTRIC POINT (pI) VALUE OF PROTEINS BY THE SIMPLIFIED METHOD
  4. DETERMINATION OF pI VALUE AND ELECTRIC CHARGES OF PROTEINS VERSUS pH USING THE IMPROVED OSCILLATING METHOD (IOM)
  5. GENERAL CONSIDERATIONS ON THE pI VALUES OF THE PROPOSED PROTEINS
  6. ON THE NOMENCLATURE OF THE ACID–BASE RESIDUES USED IN SOME TEXTBOOKS OF BIOCHEMISTRY
  7. Acknowledgements
  8. REFERENCES
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